In response to this IRCN FOA, in this application we have combined our experience of two decades in understanding the tumor microenvironment (TME) with molecular and functional imaging, and our experience of a decade in developing theranostic nanoparticles (NPs) that deliver small interfering RNA (siRNA), to understand the role of TME in siRNA NP delivery and function. siRNA have emerged as promising candidates for precision medicine in cancer that becomes significantly important for cancers such as triple negative breast cancer (TNBC) that lack targeted treatments. Here we will perform combined in vivo PET-MR imaging studies to relate siRNA NP delivery, as detected by PET imaging, to vascular parameters, acidic extracellular pH (pHe), and extracellular matrix (ECM) porosity as detected by MRI to understand the role of the TME in siRNA NP delivery and function. The dextran NP we selected is biocompatible making it an excellent translational candidate; the siRNA we selected for delivery downregulates choline kinase (Chk), an important target in cancer cells. Downregulation of Chk results in a decrease of total choline that can be imaged with MR spectroscopic imaging (MRSI), allowing us to relate siRNA delivery to function noninvasively. Both PET and MR are also easily translatable to humans. We will integrate ex vivo mass spectrometry imaging (MSI), second harmonic generation (SHG) microscopy, and immunohistochemistry (IHC) of co-localized tumor sections to expand our understanding of the changes mediated by the Chk siRNA on the proteome, lipidome and metabolome with MSI, and of the characteristics of the ECM that play a role in NP delivery and distribution with SHG microscopy and IHC. These studies will be performed with human TNBC xenografts genetically engineered to overexpress vascular endothelial growth factor (VEGF) or to report on hypoxia. The reagents, resources, tumor models and imaging technologies developed through this application will be available to the Alliance awardees and for collaborations outside of the current Alliance network to establish mutually beneficial collaborations. These studies have the potential to provide future clinically translatable applications in achieving precision medicine of cancer.